Translation initiation factors promote the assembly of 80S ribosomes at the AUG codon of an mRNA. The translation initiation factor eIF5B is a ribosome-dependent GTPase that promotes subunit joining in the final step of translation initiation. eIF5B is an ortholog of the prokaryotic translation factor IF2, and the X-ray structure of eIF5B revealed a chalice-shaped protein. Comparison of active, GTP-bound and inactive, GDP-bound eIF5B revealed that minor changes in the structure of the GTP-binding domain were amplified through a lever-type. Using an eIF5B mutant that utilizes XTP in place of GTP, we demonstrated that at least two nucleotide (GTP) hydrolysis events are required for eukaryotic translation initiation. Consistent with a role in subunit joining, yeast strains lacking eIF5B showed increased levels of leaking scanning. Mutational analysis of the GTP-binding domain of yeast eIF5B revealed a critical role for GTP-binding and hydrolysis by eIF5B for translation initiation. The Switch 1 is a conserved element in all GTP-binding proteins. Substitution of alanine for a universally conserved threonine in the Switch I of eIF5B impaired the GTPase activity of eIF5B and caused a severe slow-growth phenotype. Three intragenic suppressor mutations of this Switch I mutant restored near wild-type growth, but did not restore the GTPase activity of the factor. These suppressor mutations, which map to the ribosome-binding face of the factor, lowered the ribosome-binding affinity of eIF5B. We propose that GTP-bound eIF5B binds to 40S ribosome preinitiation complexes, where it stabilizes binding of the initiator Met-tRNA to the ribosomal P site and promotes subunit joining. Joining of the 60S ribosomal subunit triggers GTP hydrolysis by eIF5B, and coverts the factor into a form with low ribosome binding affinity. Thus, eIF5B is a regulatory GTPase in which GTP versus GDP binding governs the ribosomal affinity of the factor. The suppressor mutations uncouple eIF5B GTPase and translational activities by enabling eIF5B release from the ribosome in the absence of GTP hydrolysis. The binding of initiator methionyl-tRNA to ribosomes is catalyzed in eukaryotic organisms by the heterotrimeric factor eIF2. The kinases PKR, HRI, PERK, and GCN2 specifically phosphorylate serine-51 on the alpha subunit of eIF2 to regulate translation during stress conditions. We demonstrated that the vaccinia virus K3L and swine pox virus C8L proteins are pseudosubstrate inhibitors of PKR, and can suppress PKR function in both yeast and mammalian cells. This inhibition of PKR by K3L and C8L was dependent on residues conserved among eIF2alpha, K3L and C8L. The M156R protein from myxoma virus is a homolog of the K3L protein. We found that PKR efficiently phosphorylates the M156R protein. The M156R protein competed with eIF2alpha for phosphorylation by PKR in vitro suggesting a possible mechanism by which myxoma virus prevents PKR phosphorylation of eIF2alpha. Twelve single amino acid changes have been identified in the PKR kinase domain that render PKR resistant to inhibition by the K3L protein. These PKR mutants are currently under investigation using both in vitro kinase assays and in vivo assays of PKR function. Mutational analysis of yeast eIF2alpha revealed a stringent requirement for residues 1-180 for phosphorylation of Ser-51. Amino acid substitutions at residues 49 and 50 as well as in a conserved sequence motif around 30 residues C terminal of the Ser-51 phosphorylation site impaired translational regulation. Biochemical studies revealed that a subset of the mutations in eIF2alpha blocked phosphorylation by GCN2 and PKR both in vivo and in vitro. These results demonstrate that kinase recognition of eIF2alpha utilizes residues both nearby and, surprisingly, remote from the phosphorylation site.